This invention relates to methods for reducing the adsorption of organic materials (e.g., peptides, proteins, nucleic acids, and cells) onto hydrophobic or hydrophilic surfaces (e.g., polymeric surfaces). The invention also relates to devices, vessels and apparatus (e.g., microtiter plates, microfluidic channels and kits) having been treated by such methods and methods of performing fluid operations therein.
Biological materials such as peptides, proteins, nucleic acids, and cells are often stored, transferred or reacted in devices and apparatus such as multiwell plates, microcentrifuge tubes and pipettes made of plastic or other non-polar materials. It is a common observation that biological compounds adsorb/bind to the surfaces of such devices. This is also true for organic materials which exhibit some hydrophobicity in an aqueous solution, e.g., acridinium compounds, PCBs, etc.
For many applications, such binding is undesirable. For example, the binding results in the loss of valuable materials, such as, enzymes and antibodies, and can result in variations in the dispensing of organic materials, especially when small volumes are involved. The binding of proteins, cells, and platelets to hydrophobic surfaces is also of concern in a variety of blood handling procedures.
As a result of these considerations, extensive efforts have been made to provide methods for reducing the binding of proteins and other organic compounds to various surfaces. Examples of the approaches which have been considered can be found in Caldwell et al., U.S. Pat. No. 5,516,703; Ding et al., International Application Publication WO 94/03544; Amiji et al., Biomaterials, 13:682-692, 1992; J. Andrade, xe2x80x9cPrinciples of Protein Adsorptionxe2x80x9d in Surface and Interfacial Aspects of Biomedical Polymers, J. Andrade, editor, Volume 2, Plenum Press, New York, 1-80, 1985; Lee et al., Polymeric Mater. Sci Eng., 57:613-617, 1987; Lee et al., Journal of Biomedical Materials Research, 23:351-368, 1989; Lee et al., Biomaterials, 11:455-464, 1990; Lee et al., Prog. Polym. Sci., 20:1043-1079, 1995; Merrill et al., ASAIO Journal, 6:60-64, 1983; Okano et al., Journal of Biomedical Materials Research, 20:1035-1047, 1986; Okkema et al., J. Biomater. Sci. Polymer Edn., 1:43-62, 1989; Owens et al., Journal of Cell Science, 87:667-675, 1987; Rabinow et al., J. Biomater. Sci. Polymer Edn., 6:91-109, 1994; Schroen et al., Journal of Membrane Science, 80:265-274, 1993; Sheu et al., J. Adhesion Sci. Technol., 6:995-1009, 1992; Shinada et al., Polymer Journal, 15:649-656, 1983; and Thurow et al., Diabetologia, 27:212-218, 1984.
Of particular interest is the treatment of small volume reaction devices that allow for multiple reactions under a variety of conditions. Such advances have been made in microfluidics and microtiter plate technology, therefore, there is a need for methods of treating these devices to decrease contamination, increase reaction yields and save valuable reagents.
Microfluidics involves using microchannels instead of test tubes or microplates to carry out analyses and reactions. These microchannels or microcircuits are etched into silicon, quartz, glass, ceramics or plastic. The size of these channels is of micrometer order, while the reaction volumes are of nanolitre or of microlitre order. The principle is to guide the reaction media, which contain reagents and samples, over zones which correspond to the different steps of the protocol. The integration of reactors, chromatographic columns, capillary electrophoresis systems and miniature detection systems into these microfluidic systems allows the automation of complex protocols by integrating them into one single platform. These xe2x80x9claboratories on chipsxe2x80x9d have made it possible to obtain results which are efficient in terms of reaction speed, in terms of product economy and in terms of miniaturization which allows the development of portable devices. Remarkable results have also been obtained for the integration and automation of complex protocols, such as biochemical or molecular biology protocols which often require numerous manipulations. These manipulations comprise in particular mixing reagents and samples, controlling the reaction temperature, carrying out thermal cycling and detection. Wolley et al. (Anal. Chem., 68, 4081-4086, 1996), for example, described the integration of a PCR microreactor, a capillary electrophoresis system and a detector in a single device. A device on a chip which allows the integration of a step for mixing the reagents and an enzymatic reaction has been described by Hadd et al. (Anal. Chem., 69, 3407-3412, 1997). This device provides a microcircuit of channels and reservoirs etched into a glass substrate, and moving and mixing of the fluids takes place by electrokinetics. Numerous microfluidic systems for the integration of protocols and of analyses have thus been described in particular in international patent application WO 98/45481, the disclosure of which is incorporated herein by reference.
One of the great difficulties in implementing these devices resides in the high adsorption of samples and reagents to the channel surface during the movement of the fluids through the channel. In general, microfluidic devices contain channels in a micrometer size-range that have large surface-to-volume ratios (xcx9c10-100 times greater than a surface-to-volume ratio in conventional microtiter plates). This leads to an increased significance of the surface properties/quality/chemistry in microfluidic devices. At the same time, biological samples (e.g., protein samples, or reaction mixtures such as PCR mix, LCR mix, microsequencing (MIS) mix, etc.) are complex mixtures of large and small molecules of different polarities (e.g., DNA and protein molecules, dNTPs, ddNTPs, fluorescent labels, etc.) that may have a strong affinity to solid substrates, as well as for liquid/liquid and liquid/air interfaces. Proteins in particular are known to adsorb strongly to silica materials (Righetti, P. G., ed., 1996, Capillary Electrophoresis in Analytical Biotechnology, CRC series in Analytical Biotechnology, CRC Press, Boca Raton). For these reasons, surfaces in microfluidic devices are deactivated prior performing biological reactions in such microstructures. Deactivation of a surface reduces adsorption of organic materials onto the surface. Without deactivation of surfaces, biological reactions generally cannot be performed in silicon (silica) microchannels [Shoffner, M. A. et al., Nucleic Acids Res. 24: 375-379 (1996) and Cheng, J. et al., Nucleic Acids Res. 24: 380-385 (1996)].
Several possibilities for surface deactivation have been shown. Generally, the deactivation strategy depends on the material from which a microfluidic device is made. For example, silica surfaces (silicon chips) can be chemically functionalized, e.g., through silanisation reactions (Snyder, L. R., Kirkland, J. J., Introduction to modern chromatography, Wiley-Interscience, 1979, New York; Shoffner, M. A. et al., Nucleic Acids Res 24: 375-379 (1996); and Kopp, M. et al., Science, 280: 1046-1048 (1998)). The silanized chips can directly be used for biological reactions or may further be modified by preparation of a polymer coating layer on the surface.
Surface deactivation using coatings involves two approaches: covalent and non-covalent coatings. The stability of covalent coatings, some of which are referred to as polymer brushes, and adsorbed polymer layers depends on three factors: (a) the chemical stability of the surface, (b) the stability of the polymer-surface interaction, and (c) the chemical stability of the polymer that is used for a surface modification. Generally, with respect to the stability of a single polymer/surface interaction (a), a covalent bond (covalent coatings) is more stable than a non-covalent polymer/surface interaction (a fraction of a kBT unit, where kB is the Boltzman constant and T is temperature) in adsorbed polymer layers. However, due to a large number of segments (sometimes more than 10% of the total number of segments/monomers in a polymer) interacting with a surface in adsorbed polymer layers, the total adsorption energy per a single polymer molecule can be very large, reaching several kBT units and thus rendering the polymer adsorption virtually irreversible.
Covalent coatings have involved either growth of a polymer chain from a functionalized silica surface [Hjerten, S., J. Chromatogr., 347, 191-198 (1985); and Cobb, K. A. et al., Anal. Chem., 62: 2478-2483 (1990)] and/or grafting of a polymer chain onto a silica surface [Herren, B. J. et al., J. Colloid Interface Sci., 115:46 (1987); and Balachander, N. et al., Langmuir 6: 1621 (1990), Burns, N. L. et al., Langmuir 11:2768 (1995)]. The surface deactivation through chemical reactions involves formation of covalent linkages between functional groups on a surface (xe2x80x94OH) and a reactive silane or a functionalized polymer molecule [e.g., a silanized polyethylene glycol (PEG)]. This chemical modification of surfaces, often carried out in organic solvents, is time consuming and requires several synthetic steps before completion. Thus, this strategy of surface deactivation is generally expensive and not well adapted to modify large quantities of chips in a simple manner. The covalent chemical linkages (especially xe2x80x9cxe2x80x94Sixe2x80x94Oxe2x80x94Xxe2x80x9d bonds) are prone to a hydrolysis (e.g., at an alkaline pH) degrading the covalent coatings over time [Cobb, K. A. et al., Anal. Chem., 62, 2478-2483 (1990)]. Since these coatings cannot be simply regenerated, the life-time of such surfaces and consequently the whole devices is finite, and day-to-day reproducibility of the quality of a surface in chemically modified microfluidic devices is poor. Moreover, a good batch-to-batch reproducibility is hard to maintain due to, e.g., surface impurities and/or control of the humidity of the organic solvents used.
Another possibility for deactivating a microfluidics surface with a non-covalent coating is the adsorption of bovine serum albumin (BSA) onto a surface. BSA has been successfully employed for surface deactivations in PCR chips [Northrup, M. A., Anal. Chem., 70: 918-922 (1998); Waters, L. C. et al., Anal. Chem., 70: 158-162 (1998); and Waters, L. C. et al., Anal. Chem., 70:5172-5176 (1998)]. BSA adsorbs onto silica, saturates surface adsorption sites and enables to perform biological reactions in devices with high surface-to-volume ratios. However, BSA denatures at temperatures above 55-65xc2x0 C. and, consequently, may coagulate and form large aggregates (depending on the BSA concentration) [Wetzel, R. et al., Eur. J. Biochem. 104: 469-478 (1980); and Oakes, J., J. Chem. Soc. Faraday, I72: 228-237 (1976)]. This coagulation (denaturation) of BSA at a high temperature is irreversible, i.e., the BSA does not re-dissolve in aqueous solutions when the temperature is decreased. When biological reactions are performed in a static mode (no liquid flow), e.g., in micro-wells, the surface deactivation by BSA is robust and works well [Northrup, M. A., Anal. Chem. 70: 918-922 (1998), Waters, L. C., Anal. Chem., 70: 158-162 (1998); and Waters, L. C., Anal. Chem., 70: 5172-5176 (1998)]. However, when biological reactions are carried out in a flow (i.e., there is a non-zero shear stress at the solid/liquid interface) and in small channels, large BSA aggregates in a solution may be transported downstream and, eventually, block a microchannel or a capillary. Thus, the disadvantage of using BSA for surface deactivations in microfluidic devices where reagents are transported from one place to the other by a liquid flow is the BSA""s solubility change in a temperature range commonly used for biological reactions (PCR, LCR, MIS, etc.).
Although the use of polymers to prevent the adsorption of samples and reagents to microfluidic surfaces has been discussed thus far, the same techniques may be applied to other surfaces, such as surfaces made of silicon, quartz, glass, ceramics or plastic (polymeric), and other interfaces (i.e., liquid/liquid and liquid/air interfaces). These surfaces may include, but are not limited to, the surfaces of apparatus used for storing, dispensing, or reacting fluid samples, such as test tubes, multi-well plates, pipettes, pipette tips, microtiter plates, reaction wells or microcentrifuge tubes.
Such treatment techniques have a particular application to microtiter plates because they are used for biological reactions, often requiring successive steps in the same reaction well at small volumes. For example, genotyping process by single base extension method requires three successive biological reactions (i.e., polymerase chain reaction (PCR), enzymatic purification and microsequencing (MIS)). Thus it is efficient and beneficial to perform two or more successive reactions in the same well of one unique microtiter plate. In such cases, it must be assured that there is no inter-reaction contamination, and that biomolecules from previous reactions do not contaminate subsequent reactions. However this can prove difficult to achieve due to the high adsorption of residual biomolecules onto the surface of the microtiter plate, or high concentration of such molecules into liquid/liquid or liquid/air interfaces.
Many apparati, such as microtiter plates are non-polar, while biomolecules (e.g., protein samples, or reaction mixtures such as PCR reagents and MIS reagents) are complex mixtures of large and small molecules of different polarities (e.g., DNA and protein molecules, dNTPs, ddNTPs, fluorescent labels, etc.) that often have strong affinities to solid substrates. Adsorption of biomolecules onto the surface of the microtiter plate renders these biomolecules non-accessible or less accessible to enzymes during biological reactions.
One commonly encountered problem is adsorption of deoxynucleotides (dNTPs) onto the surface of microtiter plates, particularly during genotyping processes. Adsorption of dNTPs added during the first step of a genotyping procedure (i.e., PCR) onto the surface of a microtiter plate render them less accessible to shrimp alkaline phosphatase (SAP) during subsequent steps (i.e., enzymatic purification), and the adsorbed dNTPs are not dephosphorylated. As dNTPs release from the surface of the microtiter plate at high temperature (e.g., during the denaturation of EXO and SAP enzymes or during the temperature cycling of the MIS reactions), they can dramatically contaminate the third step of the genotyping process (i.e., MIS). As a result, the MIS oligonucleotide product may be extended by more than one base (dNTPs); thus the SNP specific fluorenscently-labeled ddNTPs may be incorrectly incorporated into the oligonucleotide product several bases downstream from the SNP site of interest. Consequently, this can lead to errors in genotyping (e.g., a homozygous sample can appear as a heterozygous one) when a detection technique without size-discrimination capability is employed. However, an additional prolongation of a MIS oligonucleotide by dNTPs left from a PCR causes a decrease of a specific signal that may be disadvantageous for any detection technique employed.
Another disadvantage related to adsorption of biomolecules onto the surface of a microtiter plate is a reduced yield of biological reactions. Absorption of valuable biomolecules results in decreased yields of the desired product. In an attempt to offset the loss of reagents to absorption, reagent concentrations are often increased to saturate the absorption sites on the plate surfaces. However, this is not an economical solution for preventing absorption.
A possibility for preventing contamination is to transfer the samples into a new microtiter plate between the two successive reactions. In the case of a genotyping process, PCR is performed in one microtiter plate and enzymatic purification followed by MIS is performed in another microtiter plate. However, this method of preventing contamination is not completely effective as about 10% of MIS reactions are still contaminated by leftover dNTPs from PCR reactions (data from Genset, Analyse Genomique Department). This contamination becomes more evident when reactions are performed in very small volumes (e.g., high-density plates), as the number of interactions of molecules from bulk solution with surfaces increases rapidly when the reaction volume decreases. Furthermore, it is technically more convenient and cheaper to use a method that does not require the transfer of samples to a new microtiter plate.
There is therefore a need for a method of deactivating surfaces which provides good day-to-day reproducibility of the quality of a surface in the same chip, microtiter plate or other surface, provides a stable surface at the relevant temperatures for biological applications, and does not involve complicated and expensive synthesis processes.
The invention provides for methods of using polymers for competitive adsorption to a surface to decrease the adsorption of organic materials (e.g., biomolecules) in fluid (e.g., aqueous) samples onto said surfaces. Alternatively, the invention provides for methods of using polymers to decrease the adsorption of organic materials onto liquid/air or liquid/liquid interfaces. Said surfaces may be made of silicon, quartz, glass, ceramics or plastic such as, e.g., polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymers, polyamides, polydimethylsiloxanes, polyurethane, polysulfone, polytetrafluoroethylene, and elastomers. These surfaces may include, but are not limited to, the surfaces of apparati such as devices or vessels used for storing, dispensing, or reacting fluid samples. Such apparati comprise, but are not limited to, test tubes, multi-well plates, pipettes, pipette tips, microtiter plates, reaction wells, microchannels, microfluidic apparati, capillaries and microcentrifuge tubes. The invention further includes methods for performing fluid operations on said surfaces such that the adsorption of organic materials in a fluid sample is minimized by the introduction of surface adsorbing polymers. Fluid operations include, but are not limited to, reactions, incubations, dilutions, titrations, purifications, detections, mixing, binding assays, drug screening assays, and measuring assays (e.g., measurement of kinetics). Also preferred are fluid operations involving an enzyme. Fluid samples include, but are not limited to, reaction mixtures, such as PCR mixtures, LCR mixtures, primer extension reaction mixtures and genotyping reaction mixtures (e.g., microsequencing mixture). Preferred fluid samples are aqueous fluid samples. Organic materials include biomolecules, such as nucleic acids (e.g., DNA, RNA, polynucleotides, oligonucleotides, nucleotides, dNTPs such as dATP, dTTP, dCTP, dGTP, and ddNTPs such as ddATP, ddTTP, ddCTP, ddGTP), amino acids (e.g., proteins polypeptides, peptides, amino acids such as asparatate, glutamate, lysine, arginine, histidine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophane, proline, serine, threonine, asparagine, glutamine, glycine, cysteine, and tyrosine) lipids, chemical compounds (e.g., fluorescent labels), and more specifically receptors or antibodies and their ligands, cells and growth factors, growth inhibitors, enzymes and substrates. Preferably, biomolecules may comprise complex mixtures of large and small molecules of different polarities such as the molecules listed above that may have a strong affinity to various surfaces. Any genius or species of surfaces, apparati, fluid operations or organic materials listed above may be specifically included or excluded from the embodiments of the invention.
The methods of the invention also relate to embodiments allowing the dynamic regeneration of surfaces where fluid samples are run sequentially in an apparatus such as a device or vessel (e.g., microchannel). Further, the methods of the invention relate to embodiments allowing for subsequent fluid operations to be performed sequentially on the same surface such that contamination is minimized and reaction yields are maximized.
Introducing surface adsorbing polymers that bind non-covalently to a surface prevents the undesired adsorption of organic materials onto the surface, thus reducing contamination and increasing reaction yields. Preferably, the surface is of smaller polarity than the polarity of an aqueous fluid sample. Such non-covalent coatings have several important advantages over chemical (covalent) coatings used in fluid operations: non-covalent deactivation methods do not involve complicated and time consuming chemical synthesis and are therefore inexpensive and well adapted to modify in a simple manner large quantities of apparatus for storing, dispensing or reacting fluid samples.
In a first aspect, the invention encompasses a method of decreasing adsorption of an organic material onto a surface, comprising: a) adding a fluid sample comprising said organic material and a surface adsorbing polymer to said surface, wherein said surface adsorbing polymer is capable of binding non-covalently to said surface. In a preferred embodiment, the surface is made of silicon, quartz, glass, ceramics or plastic (e.g., polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymers, polyamides, polydimethylsiloxanes, polyurethane, polysulfone, polytetrafluoroethylene, and elastomers). Further, the organic material is a complex mixture of large and small molecules of different polarities such as the biomolecules listed above, which may have a strong affinity to the surface of the apparatus. A fluid sample may comprise a reaction mixture, such as a PCR reaction mixture, a LCR mixture, a primer extension reaction mixture or a genotyping reaction mixture (e.g., microsequencing (MIS) mixture). In another preferred embodiment, the surface adsorbing polymer is from a family of polyacrylamides, such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA). In other preferred embodiments, other polymers, such as propylene glycol (PG) and ethylene glycol (EG), and polyglycols including polypropylene glycols (PPG) and polyethylene glycols (PEG) may be used as surface adsorbing polymers. In still other preferred embodiments, other polymers, such as propylene oxide (PO) and ethylene oxide (EO), and polyoxides including polypropylene oxides (PPO) and polyethylene oxides (PEO) may be used as surface adsorbing polymers. In further preferred embodiments, the surface adsorbing polymer is polydimethylsiloxane (PDMS) or polyvinylpyrolidone. In most preferred embodiments, block copolymers of the polymers listed herein are used, including for example block copolymers of PPG and PEG and PAM and NIPAM and PDMS. Any genus or species of surface adsorbing polymer may be specifically included or excluded from the embodiments of the invention. BSA is specifically excluded from the surface adsorbing polymers of the present invention.
In another aspect, the invention encompasses a method of decreasing adsorption of an organic material onto a surface, comprising: (a) obtaining a fluid sample comprising an organic material; (b) adding an effective amount of a surface adsorbing polymer to said fluid sample (c) contacting said fluid sample comprising said organic material and said surface adsorbing polymer to a surface; and (d) performing one or more fluid operations. Preferred fluid samples do not normally comprise any surface adsorbing polymer of the present invention prior to performing fluid operation. Alternatively, the fluid samples may normally comprise a surface adsorbing polymer of the present invention prior to performing one of the methods of the invention. Preferred fluid operations are fluid operations that do not normally involve any surface adsorbing polymer of the present invention. Other preferred fluid operations are reactions wherein said surface adsorbing polymer is not one of the reactants or wherein said surface adsorbing polymer has no activating or inhibiting effect on the reactants. Still other preferred fluid operations are reactions wherein said surface adsorbing polymer is not necessary for the reaction to occur. Alternatively, the surface adsorbing polymer may be one of the reactants of the fluid operation. Alternatively, the surface adsorbing polymer is added in excess, either in mass or molarity, compared to what is needed or normally used for performing a fluid operation. Preferably, the surface adsorbing polymer is in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, or 10,000 fold excess to what is needed to perform the reaction where the surface adsorbing polymer is a reactant or to the amount normally used where the surface adsorbing polymer is not a reactant. In a preferred embodiment, said surface is part of an apparatus that is used for storing, dispensing, or reacting fluid samples, and may include, but is not limited to, a microchannel, a test tube, a multi-well plate, a pipette, a pipette tip, a microtiter plate, a reaction well, a microchannel or a microcentrifuge tube.
In still another aspect, the invention encompasses a method of decreasing adsorption of an organic material onto the surface of an apparatus, comprising: (a) providing an apparatus comprising or consisting essentially of a surface; (b) adding a fluid sample comprising said organic material and a surface adsorbing polymer to said apparatus, wherein said surface adsorbing polymer is capable of binding non-covalently to said surface; and (c) performing one or more fluid operations in same said apparatus.
In another aspect, the invention encompasses a method of selecting the quantity of a surface adsorbing polymer that is added to a fluid operation, comprising the steps of: (a) providing an apparatus comprising consisting essentially of a surface; (b) adding a fluid sample comprising an organic material and a surface adsorbing polymer to said apparatus, wherein said surface adsorbing polymer is capable of binding non covalently to said surface; (c) performing one or more fluid operations in same said apparatus; and (d) selecting the optimum quantity of said surface adsorbing polymer capable of obtaining the highest yield. Preferably, said surface of smaller polarity than the polarity of said fluid sample. Preferably, said fluid operation is PCR.
In another aspect, the invention encompasses a method of selecting the quantity of a surface adsorbing polymer that is added to a fluid operation, comprising the steps of: (a) providing an apparatus comprising or consisting essentially of a surface; (b) adding a fluid sample comprising an organic material and a surface adsorbing polymer to said apparatus, wherein said surface adsorbing polymer is capable of binding non covalently to said surface; (c) performing one or more fluid operations in same said apparatus; and (d) selecting the optimum quantity of said surface adsorbing polymer to decrease adsorption or contamination the greatest. Optionally, said contamination of subsequent fluid operations results from the presence of undesired dNTPs due to adsorption to a surface. Preferably, said surface is of smaller polarity than the polarity of said fluid sample. Preferably, said fluid operation is microsequencing and said contamination or adsorption is detectable by the presence of sequencing artifacts.
In another aspect, the invention encompasses a kit, comprising: (a) a fluid sample for performing a fluid operation; (b) a surface adsorbing polymer in an aqueous solution; and (c) a notice recommending the quantity of said surface adsorbing polymer that should be added to the fluid sample. Optionally, said kit comprises also an apparatus consisting essentially of a surface. Optionally, said surface absorbing polymer is comprised in a reaction mixture. Preferably, the recommended quantity of said surface adsorbing polymer that should be added to the fluid sample is selected using one of the methods of the present invention. Preferably, said kit is a genotyping kit.
In still another aspect, the invention encompasses a reaction mixture for performing fluid operations, wherein said reaction mixture comprises a surface adsorbing polymer of the present invention, and wherein the quantity of said surface adsorbing polymer that is comprised in said reaction mixture was selected using the methods of the present invention. Preferably, the reaction mixture is a PCR reaction mixture, a LCR mixture, a primer extension reaction mixture or a genotyping reaction mixture (e.g., microsequencing (MIS) mixture).
In preferred embodiments, the invention encompasses a method of dynamically maintaining or regenerating the polymer coating adsorbed on the surface of a microchannel comprising: (a) providing a channel disposed in a substrate; (b) introducing to said channel at least two fluid sample zones, wherein each of said at least two fluid sample zones comprises a fluid sample of interest; and (c) providing at least one separating fluid zone located between two of said fluid sample zones, wherein said at least one separating fluid zone or at least one fluid sample zone comprises a surface adsorbing polymer capable of binding non-covalently to a surface of said channel. Optionally, at least one fluid sample zone comprises a surface adsorbing polymer. Optionally, at least one separating fluid zone comprises a surface adsorbing polymer. Optionally, at least two fluid sample zones are substantially free of said surface adsorbing polymer. Optionally, at least one fluid separating zone and at least one fluid sample zone comprises a surface adsorbing polymer. Optionally, each of said fluid sample zones comprises a surface adsorbing polymer. Optionally, each fluid separating zone comprises a surface adsorbing polymer. Optionally, each fluid separating zone and each fluid sample zone comprises a surface adsorbing polymer. Optionally, at least any one integer between 1 and 100 fluid sample zones are provided in a channel, wherein each integer may be specifically included or excluded from embodiments of the present invention. Optionally, at least one fluid sample zone and said at least one separating fluid zone are flowing in the channel.
In other aspects, the invention encompasses a method of treating the surface of a microchannel comprising: (a) providing a channel disposed in a substrate; (b) introducing to said channel a first fluid zone, wherein said first fluid zone comprises a surface adsorbing polymer capable of binding non-covalently to a surface of said channel; and (c) introducing sequentially to said channel a second fluid zone comprising a fluid sample of interest, wherein said second fluid zone is substantially free of said surface adsorbing polymer when introduced to said channel. The surface refers to the interface between the microchannel and the fluid zone. In preferred embodiments, the surface adsorbing polymer is from a family of polyacrylamides, such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA). In other preferred embodiments, other polymers, such as propylene glycol (PG) and ethylene glycol (EG), and polyglycols including polypropylene glycols (PPG) and polyethylene glycols (PEG) may be used as surface adsorbing polymers. In still other preferred embodiments, other polymers, such as propylene oxide (PO) and ethylene oxide (EO), and polyoxides including polypropylene oxides (PPO) and polyethylene oxides (PEO) may be used as surface adsorbing polymers. In further preferred embodiments, the surface adsorbing polymer is polydimethylsiloxane (PDMS) or polyvinylpyrolidone. In most preferred embodiments, block copolymers of the polymers listed herein are used, including for example block copolymers of PPG and PEG and PAM and NIPAM and PDMS, and further including block copolymers such as polyacrylamide-block-N-isopropylacrylamide (PAM-NIPAM) and polydimethylsiloxane-block-polyethyleneglycol (PDMS-PEG).
In yet further aspects, the invention encompasses a method of dynamically maintaining or regenerating the polymer coating adsorbed on the surface of a microchannel comprising: (a) providing a channel disposed in a substrate; and (b) introducing to said channel at least two fluid sample zones, wherein each of said at least two fluid sample zones comprises a fluid sample of interest, and wherein each of said at least two fluid sample zones comprises a surface adsorbing polymer capable of binding non-covalently to a surface of said channel.
In preferred embodiments of the invention, at least 2 fluid zones or fluid sample zones are provided in an individual channel. In particularly preferred embodiments, at least any one integer between 5 and 1000 fluid zones or fluid sample zones are provided in an individual channel. Preferably, adjacent fluid zones or fluid sample zones are separated by at least one fluid separating zone.
Where fluid samples and the surface adsorbing polymer are to be provided in the presence of one another, fluid samples and polymer solutions may be introduced to an apparatus of the invention either as a mixture or separately; separately introduced samples and polymers may be mixed in the apparatus.
In preferred embodiments, the polymer solution flows through the channel. Preferably, as described further, the polymer solution is moved through the channel in a continuous flow. In further preferred methods, movement of a fluid such as a solution, fluid sample zone or fluid separating zone in a channel is effected by pressure gradient. Optionally, movement of said solution is effected by an electroosmotic, electrokinetic, electro-hydrodynamic or by a temperature gradient system.
In further aspects, the invention relates to a method of performing a fluid operation in an apparatus comprising: (a) introducing a solution comprising a surface adsorbing polymer to the apparatus such that the polymer non-covalently adsorbs onto the apparatus surface; (b) introducing a fluid sample to said apparatus; and (c) performing a fluid operation in said apparatus. Preferably, said apparatus is a microtiter plate or a microfluidic apparatus.
The invention also encompasses microchannels and microfluidics devices comprising channels treated according to the methods of the invention. Specifically, the invention relates to a channel or a microfluidics device comprising a substrate and at least one channel disposed in said substrate, wherein the channel has non-covalently adsorbed thereon a surface adsorbing polymer according to the invention. Preferably, the channel has a width of between about 1 xcexcm and about 3 mm. Preferably, said substrate consists essentially of silicon, quartz, glass, ceramics or plastic such as, e.g., polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymers, polyamides, polydimethylsiloxanes, polyurethane, polysulfone, polytetrafluoroethylene, and elastomers.
Microfluidics devices of the invention may further comprise means for creating a pressure gradient across said channel, thereby effecting the movement of fluid in said channel, and/or a temperature regulation means for control of the temperature of the fluid in said channel.
The methods of the invention further comprise performing at least one fluid operation in said channel and/or said device. The methods of the invention are particularly suited to performing sequential fluid operations. The methods of the invention may comprise performing at least any one integer between 2 and 1000 fluid operations in a channel or apparatus.
In another aspect, the invention encompasses the use of a surface adsorbing polymer in a fluid sample for performing a fluid operation in a apparatus, wherein said surface adsorbing polymer is selected from the group consisting of the family of polyacrylamides, such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA). In other preferred embodiments, other polymers, such as propylene glycol (PG) and ethylene glycol (EG), and polyglycols including polypropylene glycols (PPG) and polyethylene glycols (PEG) may be used as surface adsorbing polymers. In still other preferred embodiments, other polymers, such as propylene oxide (PO) and ethylene oxide (EO), and polyoxides including polypropylene oxides (PPO) and polyethylene oxides (PEO) may be used as surface adsorbing polymers. In further preferred embodiments, the surface adsorbing polymer is polydimethylsiloxane (PDMS) or polyvinylpyrolidone. In most preferred embodiments, block copolymers of the polymers listed herein are used, including for example block copolymers of PPG and PEG and PAM and NIPAM and PDMS, and further including block copolymers such as polyacrylamide-block-N-isopropylacrylamide (PAM-NIPAM) and polydimethylsiloxane-block-polyethyleneglycol (PDMS-PEG). Optionally, said step of performing a fluid operation comprises performing a fluid operation selected from the group consisting of a reaction, incubation, dilution, titration, purification, detection and drug screening assay, binding assays, and measuring assays (e.g., measurement of kinetics).
In further preferred embodiments of the methods, systems and apparatus of the invention, a fluid sample or fluid sample of interest comprise a test analyte or reagent. More preferably, a fluid sample comprises an organic material. Preferably, said organic material is a biomolecule, wherein said biomolecule can be selected from the group consisting of: nucleic acids (e.g., DNA, RNA, polynucleotides, oligonucleotides, nucleotides, dNTPs such as dATP, dTTP, dCTP, dGTP, and ddNTPs such as ddATP, ddTTP, ddCTP, ddGTP), amino acids (e.g., polypeptides, peptides, amino acids such as asparatate, glutamate, lysine, arginine, histidine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophane, proline, serine, threonine, asparagine, glutamine, glycine, cysteine, and tyrosine), lipids, chemical compounds, and more specifically receptors or antibodies and their ligands, cells and growth factors and growth inhibitors, and enzymes and substrates. Said biomolecule may comprise complex mixtures of large and small molecules of different polarities (e.g., nucleic acids and protein molecules, dNTPs, ddNTPs, fluorescent labels, etc.) that may have a strong affinity to solid substrates. Any number of biomolecules may be included or excluded as individual species of the invention. In other aspects, a fluid sample may comprise a reaction mixture, such as a PCR reaction mixture, a LCR mixture, a primer extension reaction mixture or a genotyping reaction mixture (e.g., microsequencing (MIS) mixture), any of which may be included or excluded as species of the invention.
The methods, systems and apparatus of the invention can be used advantageously in accordance with a wide range of fluid operations, some of which are described further herein. In some aspects, the step of performing a fluid operation can comprise performing a fluid operation selected from the group consisting of a reaction, incubation, dilution, titration, purification, detection, mixing and drug screening assay, any of which may be included or excluded as species of the invention. In preferred embodiments of the invention, the fluid operation comprises performing a biochemical reaction. More specifically, a biochemical reaction may comprise primer extension reaction, temperature cycling, nucleic acid amplification reaction, or enzyme purification, any of which may be included or excluded as species of the invention. Particularly preferred temperature cycling reactions include PCR or MIS reactions. Any number or combination of fluid operations may be performed according to the methods of the invention. In one aspect, a apparatus is used for a single fluid operation, which in other aspects, at least 2 sequential fluid operations are performed in a channel or apparatus of the invention. Preferably, said sequential fluid operations are performed in the same channel or apparatus of the invention. In a preferred embodiment, the invention is directed to fluid operations in volumes of less than any integer between 20 and 0.1 xcexcl. Any integer may be specifically included or excluded from the embodiments of the present invention.
Preferred surfaces which make up apparatus of the invention are made of or consist essentially of glass, quartz, silicon, metals or plastics, any of which may be included or excluded as species of the invention. Preferably said plastic is a polymeric, and, further preferred, said polymeric is a polymer of various polarities. The surface may be polar, non-polar, hydrophilic or hydrophobic. Preferred surfaces are polar hydrophobic surfaces, non-polar hydrophobic surfaces, polar hydrophilic surfaces, and non-polar hydrophilic surfaces, any of which may be included or excluded as species of the invention. Still further preferred, the polymer is selected from the group consisting of polystyrene, polypropylene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymers, polyamides, polydimethylsiloxanes, polyurethane, polysulfone, polytetrafluoroethylene, and elastomers, any of which may be included or excluded as species of the invention. As used herein, the term substrate refers to a structural surface beneath a covering or coating (e.g., polymer coating). In the present invention, microchannels may be disposed in a substrate or apparati may be made of a substrate. In a preferred embodiment, the term apparatus is used interchangeably herein with device or vessel. Apparatus of the invention are used for storing, dispensing or reacting fluid samples. Said apparatus comprise both devices and vessels such as channels disposed in a substrate, or test tubes, multi-well plates, pipettes, pipette tips, microtiter plates, reaction wells, microchannels, microfluidic apparati, capillaries and microcentrifuge tubes test tubes, multi-well plates, pipette tips, microtiter plates, reaction wells, microcentrifuge tubes, microchannels, and the like comprising said surface, any of which may be included or excluded as species of the invention. Preferably said channel is a microchannel. Microchannels of the invention, including the microchannels disposed in the microfluidic devices of the invention, preferably have a width of between about 1 xcexcm and about 3 mm.
In certain aspects, the surface of said devices, vessels or apparatus is an untreated surface; preferably an unsilanized surface when using a silicon substrate. Said surface may be hydrophobic or hydrophilic. In other aspects, the surface may be pretreated prior to treatment with a polymer according to the invention.
The invention also relates to microfluidic devices comprising reaction wells, and to fluid operations performed in a reaction well. In one embodiment, the invention comprises moving a fluid sample through or performing a fluid operation in a channel treated according to the methods of the invention, and performing a biochemical reaction in a reaction well treated according to the methods of invention. Preferably, said reaction well is in a microtiter plate. More preferably, said microtiter plate are polypropylene microtiter plates. Microtiter plates of the invention preferably are high density microtiter plates, such as microtiter plates having 96, 384, 1536 or more wells.
In particularly preferred embodiments of the invention, the surface adsorbing polymer is a water soluble polymer or a non-polar liquid soluble polymer; in other preferred embodiments the surface adsorbing polymer is an uncharged polymer. Preferably, the polymer is a silica-adsorbing polymer. In further preferred embodiments, the polymer has a molecular weight of at least 1xc3x97103, 5xc3x97103, 1xc3x97104, 5xc3x97104, 1xc3x97105, 5xc3x97105, 1xc3x97106 or 5xc3x97106 daltons. Most preferably, the polymer has a molecular weight of at least 1xc3x97106 daltons. Preferably, the surface adsorbing polymer is selected from the family of polyacrylamides. Further preferred, said surface adsorbing polymer is selected from the family of polyacrylamides, such as polyacrylamide (PAM), N-isopropylacrylamide (NIPAM) and polydimethylacrylamide (PDMA). In other preferred embodiments, other polymers, such as propylene glycol (PG) and ethylene glycol (EG), and polyglycols including polypropylene glycols (PPG) and polyethylene glycols (PEG) may be used as surface adsorbing polymers. In still other preferred embodiments, other polymers, such as propylene oxide (PO) and ethylene oxide (EO), and polyoxides including polypropylene oxides (PPO) and polyethylene oxides (PEO) may be used as surface adsorbing polymers. In further preferred embodiments, the surface adsorbing polymer is polydimethylsiloxane (PDMS) or polyvinylpyrolidone. In most preferred embodiments, block copolymers of the polymers listed herein are used, including for example block copolymers of PPG and PEG and PAM and NIPAM and PDMS, and further including block copolymers such as polyacrylamide-block-N-isopropylacrylamide (PAM-NIPAM) and polydimethylsiloxane-block-polyethyleneglycol (PDMS-PEG), any of which may be included or excluded as species of the invention. The polymer may be in aqueous or non aqueous solution. In a most preferred embodiment, the surface adsorbing polymer has a greater affinity for the surface than the biomolecules of the fluid sample. Most preferably, the total adsorption energy per a single polymer molecule reaches at least one or more kBT units.
In addition to the methods and apparatus of the invention, the invention also encompasses compositions comprising the surface adsorbing polymers of the invention. Included, for example, as further described herein, are compositions comprising a surface adsorbing polymer according to the invention and a fluid sample of interest, wherein the surface adsorbing polymer is not one of the reactants. These compositions may, for example, be prepared and subsequently provided as a mixture to an apparatus of the invention.
The invention further comprises uses of a surface adsorbing polymer, in particular for performing a fluid operation in a microchannel or a plastic apparatus, wherein the surface adsorbing polymer is not one of the reagents of the fluid operation, or wherein said surface adsorbing polymer has no activating or inhibiting effect on the reactants, or wherein said surface adsorbing polymer is not necessary for the reaction to occur. The surface adsorbing polymer can be used as an additive in a mixture comprising a sample, e.g. on which a fluid operation is to be performed. Preferably, said fluid operation is an operation selected from the group consisting of a mixing step, a reaction, an incubation, a dilution, a titration, a detection, a drug screening assay, a binding assay, and a measuring assay (e.g., measurement of kinetics). Most preferably, the fluid operation is a biochemical reaction; more preferably, the biochemical reaction involves temperature cycling.